Formulation and method for treating skeletal muscle degeneration caused by malnutrition and disease

ABSTRACT

A formulation is provided for the treatment of the adverse affects of skeletal muscle degenerative diseases prevalent in humans in developing countries. The formulation typically includes a first component including at least one of jasmone, a jasmonate, jasmonic acid, oxo-phytodienoic acid, and a second component including at least one of an antioxidant and carnitine. Additionally, soy milk may be utilized as a delivery vehicle for the formulation for oral ingestion by a subject. The formulation is designed to replenish energy levels in disease infected muscle cells, reinstate calcium homeostasis within the muscle cells, and reduce the activity of oxidizing free radical reactions typically caused by muscle degenerative diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/863,069, entitled “Formulation and Method For TreatingSkeletal Muscle Degeneration Caused By Malnutrition and Disease”, andfiled May 22, 2001, now U.S. Pat. No. 6,465,021 issued Sep. 15, 2002,which claims priority from U.S. Provisional Patent Application Ser. No.60/205,551, entitled “Formulation and Method For Treating Human SkeletalMuscle Degenerative Diseases”, and filed May 22, 2000. The disclosuresin the above-referenced patent applications are incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to formulations for counteracting skeletalmuscle cell damage and degeneration due to the effects of malnutritionand various muscle degenerative diseases.

2. Description of the Problem

The devastating effect of malnutrition on worldwide child mortalityrates has been well documented. Indeed, statistical studies of worldwidemortality data indicates that approximately 70% of the millions ofdeaths occurring annually among children less than 5 years old indeveloping countries is associated with malnutrition singly, or incombination with diseases such as malaria, diarrhea, measles, or acuterespiratory infections. Two forms of malnutrition prevalent indeveloping African and Asian countries are kwashiorkor and marasmus.Both are forms of protein-energy malnutrition characterized by growthretardation in children and wasting of subcutaneous fat and muscle. Manyfactors add to and promote the deleterious effects of malnutritionincluding ingestion of toxins and viral infection.

Chronic ingestion of fungal mycotoxins by humans, particularlycyclopiazonic acid (CPA) and aflatoxin (AFL), contribute significantlyto the development of malnutrition and liver cancer, respectively. CPA,an indolic fungal metabolite, coexists with AFL, a liver carcinogen, innature. Assays of both CPA and AFL indicate that the two mycotoxinselicit their toxic effects by independent modes of action. The mechanismof the toxicity of CPA in mammals and its role as a very common toxicfood contaminant is now well established. CPA is a specific inhibitor ofCa-ATPase in the skeletal muscle (sarcoplasmic reticulum) of mammals.See Goeger et al., Biochemical Pharmacology, 37, 978-981 (1988).Ca-ATPase controls the pumping of calcium within the sarcoplasmicreticulum to effect muscle contraction or relaxation. Inhibition ofCa-ATPase by CPA prevents the pumping of calcium in the muscle cell thatwould otherwise be used for maintaining overall health of the cell, thusresulting in a wasting and degeneration of muscle cell tissue.

Apoptosis has also been found to occur in skeletal muscle in response toCPA-induced cell damage caused by perturbation of Ca-homeostasis andCa-ATPase. Apoptosis is an active form of cellular death and constitutesa strictly regulated device for the removal of damaged cells. Apoptosisplays a very important physiological role during organ development andis involved in disease pathogenesis. Enhanced apoptosis of cellsparticipates in chronic pathologies such as in muscle degenerativediseases. For example, an apoptotic phenomenon exists in the skeletalmuscle of experimental models of cancer cachexia, using the rat YoshidaAH-130 ascites hepatoma (liver tumor). See van Royen et al., BiochemBiophys Res Commun, 270(2): 533-537 (Apr. 13, 2000).

Another muscular-degenerative disease is hepatocellular carcinoma (HCC),which accounts for 80-90% of all liver cancers worldwide. HCC is alsoone of the ten most frequent cancers worldwide, accounting for 4% of thetotal. Major contributory factors of HCC are presumed to be fungaltoxins found in foods, such as the previously noted CPA and AFL. Theincidence of HCC is prevalent in men in African and Asian countries,infecting 4 out of every 10,000 people. There has been a strongindication that mycotoxins acting in concert with other factors such asmalnutrition, infectious diseases and the hepatitis B virus (HBV) couldbe responsible for the high incidence of HCC in Africa and Asia.

HIV also causes a form of virus-induced skeletal muscle degenerativedisease, namely, AIDS. Malnutrition is common among individualssuffering from advanced HIV-disease. HIV/AIDS malnutrition has a majorinfluence on the progression of the disease. Therefore, early andongoing nutritional and pharmacological interventions that modify and/orcontrol the dietary intake of a subject having HIV/AIDS can minimize theprogression of the disease. In fact, nutritional supplementation ofHIV/AIDS patients is necessary because it might lead to improved immunefunction. Additionally, dietary supplements with antioxidants (e.g.,vitamins C and E) at supranutritional doses have been found to protectagainst oxidative damage to skeletal muscle mitochondria caused by theantiretroviral agent AZT.

Many different classes of chemicals affect Ca-homeostasis adversely.Several of these compounds also adversely affect the redox state ofcells causing oxidative stress. It would therefore be advantageous todevelop a pharmaceutical agent or formulation that counteracts thenegative effects of skeletal muscle degenerative diseases withoutadversely affecting calcium levels or the redox state of the musclecells.

SUMMARY OF THE INVENTION

Therefore, in light of the above, and for other reasons that becomeapparent when the invention is fully described, an object of the presentinvention is to provide a formulation for preventing the degeneration ofskeletal muscle tissue commonly caused by malnutrition and degenerativediseases such HCC and AIDS.

Another object of the present invention is to provide a formulation forpreventing the degeneration of skeletal muscle tissue that does notadversely affect Ca-homeostasis or induce additional oxidative stress ofthe muscle cells.

A further object of the present invention is to provide a method ofdelivering a formulation for preventing the degeneration of skeletalmuscle tissue in subjects in an easy, efficient and substantiallynon-intrusive manner.

The aforesaid objects are achieved individually and in combination, andit is not intended that the present invention be construed as requiringtwo or more of the objects to be combined unless expressly required bythe claims attached hereto.

In accordance with the present invention, a formulation comprising atleast one of jasmone, jasmonate, jasmonic acid and oxo-phytodienoic acidis administered to a subject in an effective amount to prevent thedegeneration of skeletal muscle tissue. The formulation may also includeat least one of an antioxidant and carnitine provided in effectiveamounts to further prevent the degeneration of skeletal muscle tissues.The antioxidant preferably includes a phytoestrogen. Most preferably,the phytoestrogen includes an isoflavone such as genistein or daidzein.The formulation may further include soy milk as a delivery vehicle fororal ingestion by the subject.

The above and still further objects, features and advantages of thepresent invention will become apparent upon consideration of thefollowing detailed description. While the detailed description disclosesspecific details of the invention, it should be understood thatvariations may exist and would be apparent to those skilled in the art.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the present invention, a novel formulation is describedherein which prevents the degeneration of skeletal muscle tissue causedby malnutrition and other degenerative diseases such as HCC and AIDS. Aspreviously noted, certain mycotoxins, such as CPA and AFL, play a majorrole in protein-energy malnutrition and the degeneration of skeletalmuscle tissue. These mycotoxins are typically orally ingested viafungi-contaminated foods and are the cause of reduced Ca-ATPase activityin the muscle sarcoplasmic reticulum (SR).

Erythrocyte calcium-pumping activity and xenobiochemical studies inprotein-energy malnutrition have received a great deal of attention overthe last several years. See, for example, Okunade et al., BioscienceReports, 9, 359-68 (1984) and Thabrew et al., Xenobiotica, 12, 849-853(1982). Studies such as these have led to the conclusion that reducedCa-ATPase activity in protein-energy malnutrition may be linked with thegeneration of reactive oxygen species. Furthermore, damage to skeletalmuscle by different stresses has been found to occur by three commonpathways or reactions (McArdle et al., Basic and Applied Myology, 4,43-50 (1981)):

-   -   (i) Loss of energy supply to the cell;    -   (ii) Loss of intracellular calcium homeostasis; and    -   (iii) Over-activity of oxidizing free radical reactions.        Therefore, apart from inhibiting muscle SR Ca-ATPase activity,        CPA and AFL also induce oxidative stress and cell damage as well        as deplete cell energy. As described herein, the formulation of        the present invention will counteract the negative effects        caused by mycotoxins by specifically addressing the three        reactions associated with muscle stress and damage.

The formulation of the present invention typically comprises aneffective amount of at least one member of the jasmonate family. As usedherein, the term “jasmonate family” refers to cyclopentanones andcyclopentenones including jasmone, jasmonates such as methyl jasmonate,jasmonic acid and oxo-phytodienoic acid (OPDA). In addition, the term“effective amount” refers to an amount of a particular component in theformulation that renders the formulation effective in preventing thedegeneration of skeletal muscle tissue. Any one or combination of thesemembers of the jasmonate family is believed to be essential in theformulation of the present invention to combat the inhibition ofCa-ATPase and re-establish intracellular calcium homeostasis.

Jasmone is a natural methylpentylcyclopentenone found primarily in oilsextracted from plants such as jasmine flowers. Similarly, methyljasmonate, jasmonic acid and OPDA are also naturally derived and presentin plants and plant oils. Many cyclopentenones, particularlyprostaglandins, are used clinically in the treatment of various diseasesincluding cancer. Therefore, there is an indication that jasmone andother cyclopentenones and/or cyclopentanones of the jasmonate family,utilized at non-toxic doses, should be useful in the treatment of muscledegeneration caused by malnutrition and other diseases. Specifically,jasmone, jasmonates, jasmonic acid and OPDA, utilized alone or in anyselected combination with each other, are believed to counteract thespecific inhibition of Ca-ATPase by mycotoxins such as CPA, therebypositively regulating calcium activity in muscle cells.

While members of the jasmonate family, utilized alone or in combinationwith each other, are believed to be effective in treating the adverseeffects of skeletal muscular degenerative diseases, the formulation maybe enhanced via the addition of certain other components. Preferably,the formulation comprises an effective amount of at least one member ofthe jasmonate family combined with at least one of an antioxidant andcarnitine. Most preferably, the formulation will include effectiveamounts of at least one member of the jasmonate family, carnitine and anantioxidant.

Antioxidants are typically provided in the formulation of the presentinvention to minimize or prevent muscle wasting induced by oxidativestress. Effective antioxidants suitable for use in the present inventioninclude phytoestrogens, namely isoflavones. Preferably, a soy isoflavonesuch as genistein, daidzein or combinations thereof will be useful inthe formulation of the present invention. Genistein and daidzein eachoccur naturally and may be isolated from fermented soy products. Eachisoflavone has a positive effect in counteracting the oxidative stressto muscle cell tissue that typically occurs in muscle degenerativediseases. In particular, Genistein is a potent antioxidant capable ofreversing the deleterious action of CPA. Additionally, Genistein is aspecific inhibitor of tyrosine kinase, an extra cellularsignal-regulated kinase that is activated by oxidative stress in cells.Daidzein has similar properties as genistein and will also be effectivein counteracting reactions induced by mycotoxins leading to muscledamage. The combination of at least one member of the jasmonate familywith an antioxidant such as genistein, daidzein or combinations thereofhas a synergistic beneficial effect of establishing calcium homeostasisand reduced oxidative stress within disease infected and malnourishedmuscle cells.

Camitine, an optional third component of the present invention, isimportant for restoring energy that has been depleted in muscle cells asa result of protein-energy malnutrition and/or disease. The formulationof the present invention typically includes an effective amount ofL-carnitine, which is a naturally occurring L-isomer of carnitine thatalready exists in human body tissues and plays a crucial role in fatmetabolism and energy production. In fact, it is known that L-carnitineplays a critical role in normal skeletal muscle bioenergetics.Specifically, L-carnitine facilitates the control of beta-oxidation oflong-chain fatty acids for the restoration of energy that has been lostduring muscle damage and degeneration. Therefore, the addition ofL-carnitine in the formulation of the present invention effectivelymaintains adequate energy levels within muscle cells that wouldotherwise be depleted during muscle degeneration.

The components may be combined (e.g., mixed together, blended, agitated,etc.) in solution for administration to a subject during treatment.Preferably, an effective amount of each desired component is added to aliquid solution for oral ingestion by the subject. Soy milk will providea useful delivery vehicle for oral ingestion of the formulation due toits widespread use in developing countries such as Africa. Additionally,soy milk provides additional isoflavones that enhances the effect of theformula. However, it is noted that any suitable liquid solution (e.g.,cow's milk, water, fruit juice, etc.) may be provided that is capable ofdispersing the components of the formulation and preferably has a tastethat is desirable for oral ingestion. Additionally, it is noted that thecomponents may be combined in dry form and ingested without the use of aliquid solution.

Although the effective amount of each component may vary based upon thesubject being treated, each component will have a suitable activity whenprovided in the formulation in the following amounts: about 80-240micrograms of one or more members of the jasmonate family, about 16-24milligrams of antioxidant and about 2.4-3.6 grams of carnitine.Additionally, the components are typically immersed within an adequateamount, typically about 1 liter, of solution. Most preferably, aformulation of the present invention includes at least 100 micrograms ofone or more members of the jasmonate family, at least 20 milligrams ofantioxidant and at least 3 grams of carnitine disposed within 1 L of soymilk.

An exemplary method for preparing and administering a formulationaccording to the present invention is described as follows. One hundredmicrograms of at least one member of the jasmonate family (e.g., apharmaceutically acceptable salt of jasmone and/or methyl jasmonate) maybe combined with 20 milligrams of genistein and 3 grams of L-carnitinein 1 L of soy milk. The resultant formulation may be combined in anydesirable manner (e.g., hand mixed or mechanically blended or agitated)as desired to provide a suitable dispersion of the components in themilk. Administration of the formulation is achieved by oral ingestion ofthe entire formulation by a subject. Typically, the subject orallyingests the formulation on a daily basis. The number of days in whichthe formulation should be ingested depends upon the subject's level ofskeletal muscle degeneration and the specific cause of such degeneration(i.e., specific form of malnutrition and disease).

An exemplary method for treating subjects infected with skeletalmuscular degenerative diseases is described as follows. At least sixtyNigerian-African human subjects may be studied, wherein each subject isdiagnosed with non-cerebral severe falciparum malaria complicated withmarasmus and having a similar degree of skeletal muscular degenerationas determined by the measurement of baseline levels of serum creatinekinase (CK) and lactate dehydrogenase (LDH) in each subject. Acorrelation between muscle damage and increased levels of CK and LDH inmuscle tissue has been documented, for example, by Knitter et al., JAppl Physiol, 89(4): 1340-1344 (October 2000). At least thirty of thesubjects may be placed into a test group, with the remaining subjectsbeing placed into a control group. The test group of subjects may betreated with a daily dosage of the formulation prepared in asubstantially similar manner as described above. The formulation willcontain about 100 micrograms of at least one member of the jasmonatefamily (e.g., a pharmaceutically acceptable salt of jasmone and/ormethyl jasmonate) combined with 20 milligrams of genistein and 3 gramsof L-carnitine, wherein all of the components will be dispersed in about1 L of soy milk. The formulation may be administered to each patient ofthe test group on a daily basis for a period of about six weeks, whereasa placebo may be administered to each patient of the control groupduring the same six week period. Any additional required treatment willbe conducted in a substantially similar manner for patients in bothgroups during the six week clinical trial period. The testing proceduresfor the six week clinical trial will be carried out in accordance withthe Helsinki Declaration of 1975 as revised in 1983 and accepted by theEthical and Research Committees of Nigerian Federal Ministry of HealthNational Hospitals and the Schools of Medicine.

After the six week period, assays may be conducted of P-type ion-motiveATPases, including Ca-ATPase, on muscle biopsies of subjects in both thetest and control groups to determine differences in muscle tissue healthbetween the two groups. The specific assays may be conducted inaccordance with any conventional method for isolating and/or determiningconcentration of Ca-ATPase concentrations in human tissue. See, forexample, Everts et al., Muscle Nerve, 15(2): 162-167 (February 1992);Bababunmi et al., Biochem J., 248(1): 297-299 (Nov. 15, 1987); Bababunmiet al., Int. J. Biochem, 19(8):721-724 (1987); and Bababunmi et al.,Comp. Biochem. Physiol. B, 82(1): 117-122 (1985). The results of theassay will indicate a decrease in CK and LDH activity for subjects inthe test group in comparison to subjects in the control group, which isindicative of the prevention of further muscle degeneration in the testsubjects as a result of the formulation of the present invention.

Thus, the formulations of the present invention are useful in combatingthe multiple adverse effects of malnutrition and other degenerativediseases on muscle tissue. Specifically, the formulations have theeffect of reducing the adverse effects on muscle cells typically causedby inhibition of Ca-ATPase by certain mycotoxins (e.g., CPA and AFL).

The specific embodiments illustrated and described herein are intendedto be exemplary and not limiting on the scope of the invention. Havingdescribed specific embodiments of novel formulations and correspondingmethods for preventing skeletal muscle degeneration caused bymalnutrition and disease, it is believed that other modifications,variations and changes will be suggested to those skilled in the art inview of the teachings set forth herein. It is therefore to be understoodthat all such variations, modifications and changes are believed to fallwithin the scope of the present invention as defined by the appendedclaims.

1. A formulation comprising jasmone and carnitine.
 2. A formulationcomprising a first component selected from the group consisting of ajasmonate, jasmonic acid, oxo-phytodienoic acid and combinationsthereof, and a second component comprising carnitine.
 3. The formulationof claim 2, wherein the jasmonate includes methyl jasmonate.
 4. Theformulation of claim 2, wherein the second component further comprisesan antioxidant, and the antioxidant comprises an isoflavone.
 5. Theformulation of claim 4, wherein the antioxidant comprises a soyisoflavone selected from the group consisting of genistein, daidzein andcombinations thereof.
 6. The formulation of claim 4, wherein the amountof antioxidant in the formulation is in the range of about 16 milligramsto about 24 milligrams.
 7. The formulation of claim 2, furthercomprising soy milk.
 8. The formulation of claim 2, wherein the amountof the first component in the formulation is in the range of about 80micrograms to about 240 micrograms.
 9. The formulation of claim 2,wherein the second component includes carnitine in an amount in therange of about 2.4 grams to about 3.6 grams.
 10. The formulation ofclaim 2, wherein the second component further comprises an antioxidantand each of the first component, antioxidant and carnitine is providedin an effective amount in the formulation to prevent degeneration ofskeletal muscle tissue of a subject when the formulation is ingested bythe subject.
 11. The formulation of claim 10, wherein the amount of thefirst component in the formulation is at least about 100 milligrams, theamount of the antioxidant in the formulation is at least about 20milligrams and the amount of carnitine in the formulation is at leastabout 3 grams.
 12. A formulation comprising carnitine, an isoflavoneselected from the group consisting of genistein, daidzein andcombinations thereof, and a jasmonate family component selected from thegroup consisting of a jasmonate, jasmonic acid, oxo-phytodienoic acidand combinations thereof.
 13. The formulation of claim 12, wherein thejasmonate includes methyl jasmonate.
 14. The formulation of claim 12,wherein each of the carnitine, isoflavone and jasmonate family componentis provided in an effective amount in the formulation to preventdegeneration of skeletal muscle tissue of a subject when the formulationis ingested by the subject.
 15. The formulation of claim 12, furthercomprising soy milk.
 16. A method of manufacturing a formulationcomprising: (a) combining a selected amount of a first component with aselected amount of a second component, wherein the first component isselected from the group consisting of jasmone, a jasmonate, jasmonicacid, oxo-phytodienoic acid and combinations thereof, and the secondcomponent comprises carnitine.
 17. The method of claim 16, wherein thejasmonate includes methyl jasmonate.
 18. The method of claim 16, whereinthe second component further comprises an antioxidant, and theantioxidant comprises an isoflavone.
 19. The method of claim 18, whereinthe isoflavone includes at least one of genistein and daidzein.
 20. Themethod of claim 16, wherein the method further comprises: (b) combiningthe selected amounts of the first and second components in soy milk. 21.(Currently Amended) The method of claim 16, wherein the selected amountof the first component is at least about 100 micrograms, and the secondcomponent further comprises an antioxidant and includes at least about20 milligrams of the antioxidant and at least about 3 grams ofcarnitine.
 22. A formulation comprising soy milk and at least one of ajasmonate, jasmonic acid and oxo-phytodienoic acid.